Functionally reconstituted viral membranes containing adjuvant

ABSTRACT

Vaccines directed against antigens such as membrane proteins from pathogens or tumor cells are disclosed. Also described are methods of forming reconstituted viral membranes, with membrane fusion activity, which are lipid bilayer membranes preferably containing natural lipids of a virus, a viral fusion protein, one or more optional further antigens as well as amphiphilic adjuvants. Pharmaceutical compositions including such reconstituted viral membranes are also described.

FIELD OF THE INVENTION

The invention relates to vaccines directed against antigens such asmembrane proteins from pathogens or tumor cells. The invention furtherrelates to methods of forming reconstituted viral membranes, withmembrane fusion activity, which are lipid bilayer membranes containingthe natural lipids of a virus, amphiphilic antigens as well asamphiphilic adjuvants, and to pharmaceutical compositions comprisingsuch reconstituted viral membranes.

BACKGROUND OF THE INVENTION

Classically, vaccines against enveloped viruses either contain killed orlive attenuated viruses, or they comprise a preparation of theirconstituents (e.g. split virus or subunit preparations). Forvaccination, these preparations are usually injected. After injection,the viruses or proteins present in such vaccines are taken up byantigen-presenting cells of the immune system such as dendritic cells ormacrophages, followed by a presentation of the antigenic parts of thevaccines to effector cells of the immune system. Vaccines are effectivewhen injected because antigen-presenting cells are most abundant justunder the skin. However, it has now become clear that similar cells arealso present in the mucosa that, for instance, lines the nose (Ogra etal. 2001). In order to induce these phagocytes present in the mucosa tomount an immune response, much stronger stimulation is required than forthose present under the skin (Janeway et al. 2001).

While the injection of some viruses or proteins contained in vaccines,for example influenza or measles virus, elicits an immune response thatis sufficiently strong to protect against a later infection by the samevirus, this is not the case for many others, for example respiratorysyncytial virus. Numerous attempts to reinforce the immune response byphysical or chemical means have been undertaken. The most importantprinciples that have emerged from such experiments are: (1) for physicalstimulation, multiple copies of the viral proteins need to be combinedin particles. These particles can be whole viruses, reconstituted viralmembranes, or proteins on microparticle carriers. Particles stimulatethe immune system better than individual subunits (Ogra et al. 2001;Janeway et al. 2001). (2) Chemical stimulation on the other handrequires that the phagocytes or the effector cells of the immune systemreceive certain signals through receptors present on surface of theantigen-presenting cell, for instance through the use of adjuvants,chemical compounds that are recognized by these receptors.

With sufficient additional physicochemical stimulation, viral proteinscan elicit strong immune responses even if applied to mucous membranes,for example upon application to the nose (Ogra et al. 2001). Most of thecurrent methods and compositions for stimulating an immune response bysuch means, whether by chemical or physical means or combinations of thetwo principles, have significant disadvantages that will be outlinedbelow.

A particular kind of vaccine composition that was developed in the artis known as ‘virosomes’, which are lipid bilayers containing viralglycoproteins. Virosomes may comprise reconstituted viral membranes,generally produced by extraction of membrane proteins and lipids fromenveloped viruses with a detergent, followed by addition of lipids, andremoval of said detergent from the extracted viral membrane proteins andlipids, such that characteristic lipid bilayers are formed with theproteins protruding from them (Stegmann et al. 1987). Virosomes may alsocomprise membranes formed from purified viral proteins and synthetic ornatural lipids, or other substances that will form a bilayer. Acharacteristic feature of virosomes is that they closely mimic thecomposition, surface architecture and functional activities of thenative viral envelope. A particularly important characteristic of saidvirosomes involves the preservation of the receptor-binding and membranefusion activity of the native viral envelope, allowing the virosomes toenter the same cells that the virus would be able to enter, and to bepresented to the immune system by these same cells. Preservation ofreceptor-binding and membrane fusion activity is essential forexpression of the full immunogenic properties of said virosomes (Arkema2000; Bungener 2002).

For some viral antigens, virosomes elicit protective immune responsesthat can be strong even when the vaccine is, for example, deliveredintranasally (as is exemplified in WO 88/08718 and WO 92/19267).However, other virosome formulations exhibit only marginally improvedimmunogenicity as compared to killed virus or subunit preparations (asexemplified in (Glück et al. 1994). In this cited example, the virosomeswere generated through a protocol involving addition of exogenouslipids, which we have found to result in a composition of the virosomesand a surface architecture different from those in the native viralenvelope. It is known to a person skilled in the art that such adifferent surface architecture may affect the membrane fusion propertiesof the virosomes produced and thus their immunogenicity.

To enhance the immune response, allowing intranasal application of thisvaccine, an adjuvant protein from Escherichia coli (heat-labile toxin)was mixed with the lipid-supplemented virosome influenza vaccine (EP 0538 437). Clinical trials indicated that addition of the toxin wasabsolutely required to induce serum antibody titers equivalent toinjected vaccine (Glück et al. 1994). Although addition of the toxin didthus enhance the immunogenicity of this vaccine, it also induced aserious side effect known as Bell's Palsy, a temporary paralysis offacial muscles. Since the adjuvating effect of the toxin is due torecognition by an antigen-presenting cell, there is no certainty in thiscase that the toxin and the viral protein will contact the same cell,and therefore a relatively high concentration of the toxin will beneeded in order to ensure activation of every cell, increasing thechance that antigens will be recognized by an activated cell. Thereforethis type of virosome preparation with added lipids has a fair number ofdisadvantages.

Virosomes have also been prepared from purified influenza antigens,mixed with derivatives of muramyldipeptide (EP 0 205 098 and EP 0 487909). In this case, the muramyldipeptide derivative forms the membrane.Although muramyldipeptide is an adjuvant, and the formulation was indeedfound to enhance the immune response to the influenza antigens, muramyldipeptides are pyrogenic (Kotani et al., 1976; Dinarello et al., 1978),are cleared rapidly from the body following injection, and have localtoxicity leading to granulomas and inflammation (Ribi et al., 1979;Kohashi et al., 1980). Moreover, they have a limited shelf life atneutral pH (Powell et al., 1988), and the optimal pH to maintain theirstructural integrity is too low to allow their formulation in a vaccinetogether with the fusion protein of viruses that enter cells byreceptor-mediated endocytosis, such as the hemagglutinin of influenzavirus. Moreover, such synthetic membranes are not a good mimic of thenatural viral membrane and thus the immune response to them will differfrom that generated against the virus.

Alternatively, researchers in the art have also generated complexedantigens different from reconstituted viral membranes, such as‘Immunostimulatory Complexes’ (ISCOMs, Morein et al. 1984), containingviral proteins complexed with adjuvants such as saponins like Quil A®(EP 0231039B1; EP 0109942A1; EP 0180564A1), most of which are isolatedfrom the bark of Quillaia sopanaria Molina. Mixed with antigen, andlipids such as cholesterol, these adjuvants form cage-like structures ofbetween 30-40 nm, rendering the antigen particulate, while acting at thesame time as an adjuvant. Although ISCOMs have been used in a number ofveterinary vaccines, and enhance the immunogenicity of the viralmembrane proteins, the development of such vaccines for humans has beeninhibited by concerns about their toxicity and the complexity of themixture (Cox et al. 1998).

More recently, proteosome influenza vaccines were developed (USapplication 20010053368), consisting of non-covalent complexes of thepurified outer membrane proteins of bacteria such as meningococci, mixedwith antigenic proteins such as the influenza hemagglutinin or the humanimmunodeficiency envelope glycoprotein. While these multiple bacterialproteins may act as adjuvants, the complex nature of such mixtures,consisting of multiple proteins, will present a regulatory issue.Moreover, the immune response is directed against all of the proteinsand other antigens present in the solution, and less specificallyagainst the viral proteins.

Another particulate formulation developed by Biovector Therapeuticsconsists of an inner core of carbohydrate surrounded by a lipid envelopecontaining antigens. With influenza hemagglutinin as the antigen, someenhancement of the immune response was noted, but not significant enoughto warrant further development.

Live attenuated versions of respiratory viruses, such as a cold-adaptedstrain of influenza virus with minimal replication in the respiratorytract have been developed as intranasal vaccines. These vaccines havethe distinct advantage of inducing immune responses that are close tothe natural immunity induced by an infection with wild-type virus. Forinfluenza, such vaccines have been known since the 1980's, and nowappear close to commercialization. The delay has been caused by theability, that many viruses share, to mutate rapidly, causing theattenuated viruses to revert partially of wholly to wild-type virus, andthereby in fact causing the disease they were meant to prevent.

For the above reasons, it is well recognized in the art that, especiallyto induce immune responses for pathogens that do not by themselvesinduce a strong immune response, and for intranasal and other mucosalapplications, although compositions such as ISCOM's and proteosomes weredeveloped, there still is a great need for well characterized vaccinecompositions that induce a strong immune response, do not contain livevirus, and have a low toxicity.

SUMMARY OF THE INVENTION

The present invention provides novel means and methods that solve anumber of problems and difficulties outlined above. The inventionprovides a reconstituted viral membrane comprising an amphiphilicadjuvant and an antigen, wherein said adjuvant and said antigen interactthrough hydrophobic interactions, are both present with the lipidbilayer membrane of the reconstituted viral membranes, and in which thereconstituted viral membrane has membrane fusion activity that issuperior to that of virosomes prepared according to EP 0 538 437. Thereconstituted viral membrane further closely mimics the composition,surface architecture and functional properties of the viral envelopefrom which the reconstituted viral membrane is derived. The inventionfurther provides a method for producing such reconstituted viralmembranes, comprising some or all of the following steps: i) dissolvingthe virus in a suitable detergent ii) removing the viral geneticmaterial and core proteins iii) contacting one or more amphiphilicmolecules having adjuvant activity and an antigen in a solutioncomprising a detergent; and iv) removing the detergent under conditionsthat allow reformation of the membrane.

Moreover, the invention provides a pharmaceutical preparation comprisingreconstituted viral membranes according to the invention, apharmaceutically acceptable carrier, as well as the use of suchreconstituted viral membranes or a pharmaceutical preparation accordingto the invention in therapy or prophylaxis, either by intranasal, oralor parenteral delivery.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention pertains to a reconstitutedviral membrane. The reconstituted viral membrane preferably comprises:(a) a lipid bilayer; (b) a fusion protein of a virus; (c) an amphiphilicadjuvant; and, (d) optionally, a further antigen. In the reconstitutedviral membrane, preferably, the lipid bilayer has a lipid compositionthat is compatible with fusion, as induced by the fusion protein, of theviral membrane with a host cell of a natural host of the virus.Preferably lipid composition is compatible with fusion at the optimal pHof fusion. Preferably, the fusion protein, the amphiphilic adjuvant andpreferably also the optional further antigen interact with thehydrophobic interior of the lipid bilayer, i.e. are associated with,integrated into, and/or embedded in the bilayer of the viral membranethrough hydrophobic interactions with the lipids of the bilayer and/oreach other. Further preferred is that the fusion protein and theamphiphilic adjuvant are not covalently linked. Preferably, theamphiphilic adjuvant and the further antigen are also not covalentlylinked. The viral membranes of the invention are preferably functionallyreconstituted viral membranes comprising lipids, preferably naturallipids of a virus, an amphiphilic adjuvant, a viral fusion protein andone or more antigens, wherein the amphiphilic adjuvant, lipids viralfusion proteins and antigens interact primarily through hydrophobicinteractions, wherein the hydrophobic part of the amphiphilic adjuvantpreferably forms an integral part of a lipid bilayer membrane, whichbilayer further contains the fusion protein, antigen(s) and lipids. Byfunctional reconstitution is meant, that the reconstituted membrane hasmembrane fusion activity. A preferred reconstituted viral membrane is inthe form of a vesicle.

A fusion protein of a virus of a virus is herein understood to mean anintegral membrane protein of a virus, usually an enveloped virus that,if expressed on the surface of a suitable mammalian (or avian) cell, caninduce fusion of the cell, at an appropriate pH, with cells that are anatural host for the virus (see e.g. Hernandez et al., 1996). Examplesof viral fusion proteins for incorporation into the reconstituted viralmembrane include the Semliki Forest virus E1 protein, the Influenzavirus hemagglutinin (HA) protein, the HIV gp120/gp41 proteins, the Fproteins of paramyxoviruses. Two types of viral fusion protein inducedfusion can be distinguished. The first type of fusion, such as e.g.induced by the HIV gp120/gp41 proteins, occurs at neutral pH at thesurface of the targeted host cell. The second type of fusion, such ase.g. induced by the Influenza virus hemagglutinin (HA) protein, occursupon internalization at lower pH (5.0-6.5) from within the endosomalcompartment of the host cell. Both types of fusion are specificallyincluded in the present invention.

The capability of the reconstituted viral membranes of the invention tofuse with a host cell is thus dependent on the presence of anappropriate viral fusion protein. However, this capability is furtherdependent of the lipid composition of the bilayer of the reconstitutedviral membrane, as virosomes composed of synthetic lipids and viralfusion proteins have been described in the art that are incapable offusion. The lipid composition of the reconstituted viral membranes isthus preferably chosen such that the membranes are capable of fusionwith appropriate host cells at an appropriate pH. The capability of thereconstituted viral membranes to fuse may be assayed in an erythrocyteghost fusion assay as e.g. described in Example 3 herein. Forreconstituted viral membranes comprising the influenza hemagglutinin, apreferred fusion activity in this assay induces the fusion of at least30% of reconstituted viral membrane vesicles with erythrocyte ghostsafter 1 minute, if 1 μM virosomes is mixed with 50 μM erythrocyte ghostsmembrane phospholipid at a pH that is optimal for the hemagglutinin inquestion.

A preferred fusion activity for other reconstituted viral membranes,that cannot be tested by the above assay, is the fusion upon addition ofthe reconstituted viral membranes to cells capable of being infected bythe virus from which their fusion proteins are derived. Thereconstituted membranes should fuse at least 10% of the cells that wouldbe fused by the virus from which their fusion proteins are derived.

One preferred lipid composition that provides the reconstituted viralmembranes with fusion activity is a lipid composition that comprisesnatural lipids of a virus. The term “natural lipids of a virus” isherein understood to mean those lipids that are present in the membraneof a virus grown on cells, preferably mammalian, or grown on embryonatedeggs. The natural lipids of a virus are thus preferably obtained orisolated from virus particles thus grown, as opposed to syntheticlipids. However, functionally reconstituted viral membranes of theinvention may comprise purified lipids from other sources, e.g.synthetic lipids, in addition to the natural lipids. A lipid compositionfor the provision of the reconstituted viral membranes with fusionactivity is thus preferably a composition that is obtained or obtainablefrom natural viral membranes. Lipid compositions for use in the presentinvention thus include compositions exclusively composed of naturallipids of a virus, compositions composed of natural lipids of a virussupplemented with lipids from other sources, as well as compositionscomposed of lipids from various sources, which mimic the lipidcomposition of a natural viral membrane.

Adjuvants are herein intended to include any substance or compound that,when used, in combination with an antigen, to immunise a human or ananimal, stimulates the immune system, thereby provoking, enhancing orfacilitating the immune response against the antigen, preferably withoutgenerating a specific immune response to the adjuvant itself. Preferredadjuvants enhance the immune response against a given antigen by atleast a factor of 1.5, 2, 2.5, 5, 10 or 20, as compared to the immuneresponse generated against the antigen under the same conditions but inthe absence of the adjuvant. Tests for determining the statisticalaverage enhancement of the immune response against a given antigen asproduced by an adjuvant in a group of animals or humans over acorresponding control group are available in the art. The adjuvantpreferably is capable of enhancing the immune response against at leasttwo different antigens. The adjuvant of the invention will usually be acompound that is foreign to a mammal, thereby excludingimmunostimulatory compounds that are endogenous to mammals, such as e.g.interleukins, interferons and other hormones. The adjuvants to beincorporated in the functionally reconstituted viral membranes of theinvention are preferably amphiphilic adjuvants.

The term “amphiphilic adjuvant” is intended to include any adjuvant,including compounds like lipopeptides and glycolipids, havinghydrophobic membrane embedded and environment oriented polar (headgroup) moieties and which, preferably by itself, can associate with, ormore preferably integrate into lipid bilayer vesicles or micelles inwater. The term also includes any amphiphilic adjuvant that is stablyincorporated into lipid bilayers (comprising the natural lipids of avirus) with its hydrophobic moiety in contact with the interior,hydrophobic region of the bilayer membrane, and its polar head groupmoiety oriented toward the exterior, polar surface of the membrane.However, more hydrophobic adjuvants having a less pronouncedamphiphilicity, i.e. having no or only weakly polar head group moieties,but which can associate with, or integrate into lipid bilayer vesicles,are specifically not excluded from the invention. The “amphiphilicadjuvants” with adjuvant activity as used herein, thus include naturallyoccurring or (partly) synthetic adjuvants that are capable of forming areconstituted viral membrane together with one or more antigens ofinterest and natural lipids of a virus in an aqueous environment underconditions that allow the formation of a reconstituted viral membrane.

In a preferred embodiment, the amphiphilic adjuvant present in thereconstituted viral membrane is pharmaceutically acceptable for use inhumans, in contrast to e.g. Quil A™ or other saponins, which areamphiphiles with adjuvant activity that have been tested in certainsettings in the art. The amphiphilic adjuvants of the invention arepreferably not covalently linked to the antigens but are presenttogether in the lipid bilayer of the reconstituted membrane. The factthe antigen and adjuvant are not covalently linked assures thatprocessing of the antigen and presentation of its epitopes to the immunesystem is essentially identical to that of the natural protein alone,ensuring good recognition of the protein present on the naturalpathogen. On the other hand, the hydrophobic interaction of the antigenand the adjuvant with the lipid bilayer (and each other) allows for adistribution of the adjuvant and antigen over the reconstituted viralmembranes in a preparation whereby the majority of the membrane vesiclesin a preparation contain both the antigen and adjuvant in a singlevesicle, more preferably at least 60, 70, 80, 90, 95 or 95% of thevesicles contain both the antigen and adjuvant. The combination ofantigen and adjuvant in a single membrane or vesicle allows delivery ofthe antigen to the antigen presenting cell that is activated by theadjuvant, thereby increasing the therapeutic and/or prophylacticefficacy of the reconstituted viral membranes.

In a preferred embodiment of the invention said amphiphilic adjuvant isrecognized by a Toll-like-receptor (TLR) present on antigen presentingcells. Various compounds recognized by TLR's are known in the art andinclude e.g. lipopeptides, lipopolysaccharides, peptidoglycans,liopteichoic acids, lipoproteins (from mycoplasma, mycobacteria orspirochetes), double-stranded RNA (poly I:C), unmethylated DNA,lipoarabinomannan, flagellin, CpG-containing DNA, and imidazoquinolines.Not all TLR-recognised compounds are suitable as adjuvants as e.g. thetoxicity of wild-type Gram-negative bacterial lipopolysaccharides is toohigh for them to be used as adjuvants, i.e. they are notpharmaceutically acceptable for use in humans. The other TLR-recognisedcompounds may however be used as adjuvants. Such TLR-recognizedadjuvants may be amphiphilic adjuvants by themselves, or alternativelythey may be modified into an amphiphilic adjuvant, e.g. by couplinghydrophobic compounds (see below) to a polar TLR ligand. Alternatively,the amphiphilic adjuvants may target other receptors. A preferredamphiphilic adjuvant is a lipopeptide, which may be producedsynthetically or semi-synthetically. A preferred lipopeptide for use asamphiphilic adjuvant has adjuvant activity and is pharmaceuticallyacceptable for use in humans. A lipopeptide of the invention is amolecule that will usually consist of one or more (oligo)peptidescovalently coupled to one or more hydrophobic compounds selected fromfatty acids, lipids, ceramides, plasmalogens, alkyl or alkene chains, orsterols. Generally, lipopeptides for use in the present inventionpreferably comprise 3, 4, 5, 6, 7, or 8, amino acids, preferably thepeptides comprise 40-70% amino acids that are positively charged, ofwhich lysine and arginine are preferred, and preferably the peptidescomprises one or more serines and/or cysteines. Especially preferredlipopeptides are listed in Table 1.

In another embodiment of the invention said amphiphilic adjuvant is aglycolipid. A preferred glycolipid for use as amphiphilic adjuvant hasadjuvant activity and is pharmaceutically acceptable for use in humans.Glycolipids are lipids (or other hydrophobic compounds) covalentlycoupled to one or more sugars. In a highly preferred embodiment theinvention provides reconstituted viral membranes according to theinvention, in which the glycolipid is a α-galactosylceramide or aphosphatidyl inositol mannoside. The terms “an α-galactosylceramide” and“a phosphatidyl inositol mannoside” are intended to include anyderivative of either one. Derivatives of these molecules having adjuvantactivity and that are useful in the context of the present invention aree.g. described in U.S. Pat. No. 5,936,076 and in U.S. Pat. No.4,542,212, respectively. Other suitable glycolipid adjuvants for use inthe invention include e.g. modified forms of endotoxiclipopolysaccharides (LPS) of Gram-negative bacteria having reducedtoxicity of the Lipid A portion the LPS but retaining (part of) theadjuvant activity, as may be obtained from genetically modified Gramnegative pathogens and as reviewed in WO02/09746.

A modified LPS for use as amphiphilic adjuvant in the inventionpreferably has a modified Lipid A moiety with reduced toxicity. Thetoxicity of a modified LPS preferably is less than the toxicity of acorresponding wild-type LPS, more preferably the toxicity of themodified LPS is less than 90, 80, 60, 40, 20, 10, 5, 2, 1, 0.5 or 0.2%of the toxicity of the wild-type LPS. The toxicities of wild-type andvarious modified LPS's with reduced toxicity may be determined in anysuitable assay known in the art. A preferred assay for determining thetoxicity, i.e. the biological activity of the modified LPS's is the WEHItest for TNF-alpha induction in the MM6 macrophage cell line (Espevikand Niessen, 1986, J. Immunol. Methods 95: 99-105; Ziegler-Heitbrock etal., 1988, Int. J. Cancer 41: 456-461). On the other hand, a modifiedLPS with reduced toxicity should still have sufficient immunostimulatoryactivity, i.e. adjuvant activity. The modified LPS with reduced toxicitypreferably has at least 10, 20, 40, 80, 90 or 100% of theimmunostimulatory activity of the corresponding wild-type LPS. Theimmunostimulatory activity may be determined in vivo in laboratoryanimals as described above or in the Examples herein, or in vitro, e.g.determining the maturation of dendritic cells stimulated by incubationwith the LPS to be tested by measuring the production of at least onecytokine (e.g. one of IL12, IL10, INF-alpha, IL6 and IL-1-beta) by theLPS-stimulated dendritic cells, or by measuring the expression of atleast one costimulatory molecule (e.g. CD40 or CD86) on theLPS-stimulated dendritic cells.

In another aspect of the present invention, the amphiphilic adjuvantpresent in the virosome according to the invention, is a peptide,preferably an amphiphilic peptide. A preferred peptide for use asamphiphilic adjuvant has adjuvant activity and is pharmaceuticallyacceptable for use in humans. Peptides, in particular polar peptides,with adjuvant activity may be rendered into amphiphilic adjuvants by(covalently) linking them to a suitable hydrophobic compound (seeabove). Alternatively, amphiphilic peptides may comprise a hydrophobicstretch of amino acids such as a transmembrane sequence as describedbelow. A preferred peptide comprises a sequence from the Notch ligandJagged-1 (see Weijzen et al., 2002; Genbank accession no. AAC 52020) ora sequence from the Staphylococcus aureus protein A. Peptides havingsequences from Jagged-1 or protein A are preferably covalently coupledto a suitable hydrophobic compound (see above) and/or comprise atransmembrane sequence (see below). The (polar) part of the Jagged-1 orprotein A derived peptides that protrudes from the lipid bilayerpreferably comprises no more than 3, 4, 5, 6, 7, or 8, amino acids.

The reconstituted viral membranes of the invention are preferablysuitable for both parenteral and mucosal (e.g. intranasal or oral)administration. An important aspect of the present invention is,however, that the reconstituted viral membranes of the present inventioncan be applied for intranasal delivery of antigens that would notnormally elicit a sufficient immune response upon intranasal delivery inthe treated subject to protect against subsequent infection by thepathogenic organism comprising the antigen.

The reconstituted viral membranes of the invention comprise a viralfusion protein and, optionally a further antigen. Thus, it is to beunderstood that the reconstituted viral membranes comprising only aviral fusion protein and no further antigens are a part of theinvention, in which case the viral fusion protein also has a function asantigen, in addition to its function as fusion protein. On the otherhand, the reconstituted viral membranes may thus comprise one or morefurther antigens in addition to the viral fusion protein.

The antigens that are part of the reconstituted viral membrane accordingto the invention preferably have a hydrophobic part that is capable ofbeing inserted in the lipid bilayer membrane of the reconstituted viralmembrane vesicle. Many pathogenic entities such as viruses, bacteria,yeasts and parasites carry in their capsid, cell wall or membrane,proteins that elicit an immune response in the host. Examples ofantigens that have hydrophobic elements, such as e.g. transmembranesegments, and that are suited to be part of a reconstituted viralmembrane according to the invention are proteins present in the membrane(also called envelope in the case of viruses) of the pathogen.Therefore, in preferably, the antigen present in the reconstituted viralmembrane of the invention is an integral membrane protein. The antigenicproteins in the reconstituted viral membranes of the present inventionare oriented in the same way as they appear on the viral or cellularmembrane, but may present epitopes that are normally partially or atleast temporarily hidden when present in a membrane lipid bilayer.Stimulation of the immune system by these antigen-presentingreconstituted viral membranes may be due to a combination of theirspecific recognition by cells of the immune system, their particularcharacter, the presentation of the protein, and the uncovering of hiddenepitopes. Preferably, the antigenic proteins that are used in thereconstituted viral membranes of the invention comprise one or moreprotective epitopes, i.e. epitopes capable of eliciting an immuneresponse in a mammal that provides protection against infection by thepathogen from which the antigen is derived, or that provides protectionagainst a tumor expressing the antigen.

In preferred embodiments, said antigens are derived from a virus, aparasite, a fungus or a bacterium. Especially preferred arereconstituted viral membranes, wherein said antigen is derived frominfluenza virus. Proteins from influenza virus that can be used inreconstituted viral membranes of the present invention are preferablythe hemagglutinin (HA) protein, the neuramidase (NA) protein and/or theM2 protein, alone or in combination.

Antigens that can be applied and used in the formation of thereconstituted viral membranes according to the invention can be derivedfrom all sorts of viruses, non-limiting examples of such viruses are:Retroviridae such as Human Immunodeficiency virus (HIV); a rubellavirus;paramyxoviridae such as parainfluenza viruses, measles, mumps,respiratory syncytial virus, human metapneumovirus; flaviviridae such asyellow fever virus, dengue virus, Hepatitis C Virus (HCV), JapaneseEncephalitis Virus (JEV), tick-borne encephalitis, St. Louisencephalitis or West Nile virus; Herpesviridae such as Herpes Simplexvirus, cytomegalovirus, Epstein-Barr virus; Bunyaviridae; Arenaviridae;Hantaviridae such as Hantaan; Coronaviridae; Papovaviridae such as humanPapillomavirus; Rhabdoviridae such as rabies virus. Coronaviridae suchas human coronavirus; Alphaviridae, Arteriviridae, filoviridae such asEbolavirus, Arenaviridae, poxyiridae such as smallpox virus, and Africanswine fever virus. Likewise such antigens may be derived from pathogenicbacteria, fungi (including yeasts), or parasites. Such antigens includebacterial antigens of e.g. Helicobacter, such as H. pylori, Neisseria,such as N. mengitidis, Haemophilus, such as H. influenza, Bordetella,such as B. pertussis, Chlamydia, Streptococcus, such as Streptococcussp. serotype A, Vibrio, such as V. cholera, Gram-negative entericpathogens including e.g. Salmonella, Shigella, Campylobacter andEscherichia, as well as antigen from bacteria causing anthrax, leprosy,tuberculosis, diphtheria, Lyme disease, syphilis, typhoid fever, andgonorrhea. Antigens from parasites e.g. include antigens fromprotozoans, such as Babeosis bovis, Plasmodium, Leishmania spp.Toxoplasma gondii, and Trypanosoma, such as T. cruzi. Fungal antigensmay include antigens from fungi such as Aspergillus sp., Candidaalbicans, Cryptococcus, such as e.g C. neoformans, and Histoplasmacapsulatum.

Although vaccination is generally applied for the prophylacticprotection against pathogens or for the treatment of diseases followingpathogenic infection, the person skilled in the art is aware of theapplication of vaccines for tumor-treatment. Moreover, an increasingnumber of tumor-specific proteins are found to be proper entities thatcan be targeted by human or humanized antibodies. Such tumor-specificproteins are also within the scope of the present invention. Many tumorspecific antigens are known in the art. Therefore, in one preferredembodiment, the present invention provides reconstituted viral membranescomprising a tumor-specific antigen. Suitable tumor antigens includee.g. carcinoembryonic antigen, prostate-specific membrane antigen,prostate specific antigen, protein MZ2-E, polymorphic epithelial mucin(PEM), folate-binding-protein LK26, truncated epidermal growth factorreceptor (EGRF), Thomsen-Friedenreich (T) antigen, GM-2 and GD-2gangliosides, Ep-CAM, mucin-1, epithialial glycoprotein-2, and colonspecific antigen.

Preferred antigens from these pathogens are integral membrane proteins.However, non-membrane protein antigens or parts thereof containingprotective epitopes may also be modified for use in the presentinvention fusing them to a transmembrane sequence. Transmembranesequences or membrane-anchoring sequences are well known in the art andare based on the genetic geometry of mammalian transmembrane molecules.A transmembrane sequence usually consists of a stretch of about 10-30,usually around 20 amino acids, the majority of which having hydrophobicside chains. Transmembrane sequences are known for a wide variety ofproteins and any of these may be used. Examples of membrane-anchoringsequences for use in the present invention include e.g. those derivedfrom CD8, ICAM-2, IL-8R, CD4 and LFA-1. Preferably a transmembranesequence is derived from viral integral membrane protein that isnaturally present in a viral membrane. Examples thereof include thetransmembrane region of human respiratory syncytial virus (RSV)glycoprotein G (e.g. amino acids 38 to 63) or the transmembrane regionof influenza virus neuraminidase (e.g. amino acids 7 to 27).

In another aspect, the present invention provides a method for producinga reconstituted viral membrane, comprising some or all of the followingsteps: (a) mixing an amphiphilic adjuvant, a viral fusion protein, anoptional further antigen, and lipids in a solution comprising adetergent; (b) decreasing the concentration of the detergent underconditions that allow reconstitution of a viral membrane comprising alipid bilayer in which the amphiphilic adjuvant and the viral fusionprotein interact with the hydrophobic interior of the lipid bilayer,whereby preferably the amphiphilic adjuvant and the viral fusion proteinare not covalently linked, whereby preferably also the amphiphilicadjuvant and the optional further antigen are not covalently linked, andwhereby the reconstituted viral membrane has membrane fusion activity;(c) optionally, purifying the reconstituted viral membrane; and, (d)optionally, formulating the reconstituted viral membrane into apharmaceutical composition. For the provision of viral lipids the methodmay further comprise: i) dissolving the virus in a suitable detergentsuch as octaethyleneglycol mono-N-dodecylether ii) removing the viralgenetic material and core proteins e.g. by differentialultracentrifugation The detergent concentration is preferably decreasedby dialysis, diafiltration or absorption onto hydrophobic (and/or intosize exclusion) beads, at the appropriate rate of removal of thedetergent, that allows reformation of the membrane, wherein preferablythe amphiphilic adjuvant and the viral fusion protein and preferablyalso the further antigen, present in said reconstituted viral membrane,interact through hydrophobic interactions, with the interior,hydrophobic region of the bilayer membrane, and/or with each other. Thevirus preferably is a membrane-containing virus such as most envelopedviruses. Preferred viruses for use as source of natural viral lipids areinfluenza viruses, Semliki Forest virus, or paramyxoviruses.

Preferably, the method for producing a reconstituted viral membranedisclosed by the present invention comprises the step of purifying saidreconstituted viral membrane. Methods for purification of reconstitutedviral membranes are known in the art and include e.g. differential anddensity gradient centrifugation and/or chromatography (size exclusion-,ion exchange- and/or affinity-chromatography). Detergents areamphiphilic molecules with surface activity Suitable detergents aredetergent that efficiently dissolve the viral membrane components, butthat do not denature the fusion protein, viral capsid and/or coreproteins, e.g. zwitterionic detergents such as octaethyleneglycolmono-N-dodecylether.

Hydrophobic interactions result from non-covalent, non-electrostaticattraction forces between hydrophobic substances that are present in anaqueous environment. In a further aspect the present invention providesa pharmaceutical preparation comprising as active ingredient areconstituted viral membrane according to the invention, and apharmaceutically acceptable carrier. Pharmaceutically acceptablestabilizing agents, osmotic agents, buffering agents, dispersing agents,and the like may also be incorporated into the pharmaceuticalcompositions. The preferred form depends on the intended mode ofadministration and therapeutic application. The pharmaceutical carriercan be any compatible, non-toxic substance suitable to deliver thereconstituted viral membranes to the patient. Pharmaceuticallyacceptable carriers for intranasal delivery are exemplified by water,buffered saline solutions, glycerin, polysorbate 20, cremophor EL, andan aqueous mixture of caprylic/capric glyceride, and may be buffered toprovide a neutral pH environment. Pharmaceutically acceptable carriersfor parenteral delivery are exemplified by sterile buffered 0.9% NaCl or5% glucose optionally supplemented with a 20% albumin. Preparations forparental administration must be sterile. The parental route foradministration of the polypeptide or antibody is in accord with knownmethods, e.g. injection or infusion by intravenous, intraperitoneal,intramuscular, intrarterial or intralesional routes. The reconstitutedviral membranes are preferably administered by bolus injection. Atypical pharmaceutical composition for intramuscular injection would bemade up to contain, for example, 1-10 ml of phosphate buffered salineand 1 to 100 μg, preferably 15-45 μg (of antigen protein) of thereconstituted viral membranes of the present invention. For oraladministration, the active ingredient can be administered in liquiddosage forms, such as elixirs, syrups, and suspensions. Liquid dosageforms for oral administration can contain coloring and flavoring toincrease patient acceptance. Methods for preparing parenterally, orallyor intranasally administrable compositions are well known in the art anddescribed in more detail in various sources, including, for example,Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton,Pa., 1980) (incorporated by reference in its entirety for all purposes).In a further aspect, the invention relates to a method for vaccinationagainst, or for prophylaxis or therapy of an infectious disease or tumorby administration of a therapeutically or prophylactically effectiveamount of (a pharmaceutical composition comprising) reconstituted viralmembranes of the invention to a subject in need of prophylaxis ortherapy. The invention also relates to reconstituted viral membranes ofthe invention for use as a medicament, preferably a medicament forvaccination against, or for prophylaxis or therapy of an infectiousdisease or tumor. The invention further relates to the use ofreconstituted viral membranes of the invention in the manufacture of amedicament for vaccination against, or for prophylaxis or therapy of aninfectious disease or tumor.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic drawing of the sucrose gradient used to analyze thephysical association between lipopeptides, protein and lipids of theadjuvant-containing reconstituted viral membranes.

FIG. 2: Two-dimensional thin layer chromatogram of the lipids andlipopeptides recovered from the 10/40% sucrose interface on gradients asoutlined in FIG. 1. Panel A: control, reconstituted viral membraneswithout lipopeptide, showing the ninhydrin-reactive natural virallipids. Panel B: reconstituted viral membranes containing lipopeptides,showing the natural viral lipids reactive with ninhydrin, and theninhydrin-reactive lipopeptides in addition. The chromatograms weredeveloped in two dimensions: system 1 CHCl₃/methanol/H₂O 65/25/4, system2 N-butanol/acetic acid/water 2/1/1, and stained by derivatization withninhydrin stain. Sample loading sites are marked “spot”.

FIG. 3: Electron micrograph of reconstituted viral membranes containinglipopeptides according to the present invention; negative stain usingammonium phosphomolybdate. The membranes are about 100-200 nm indiameter.

FIG. 4: IgA titers in nose and lung after two intranasal vaccinationswith A/Panama/2007/99, 14 days apart; the titers were determined 3 weeksafter the last vaccination. Pre-immune titers were subtracted. Vaccinesused were a standard commercial subunit vaccine, virosomes preparedaccording to EP 0538437, or reconstituted viral membranes, containinglipopeptides, according to the present invention. Group size is 10 mice.

FIG. 5: IgG titers in blood after two intranasal vaccinations, 14 daysapart; the titers were determined 3 weeks after the last vaccination.Pre-immune titers were subtracted. Vaccines used were virosomes preparedaccording to EP 0538437, or reconstituted viral membranes, containinglipopeptides, according to the present invention. Four different vaccinepreparations, each containing antigen from one strain of virus asindicated, were used to vaccinate 4 groups of 10 mice.

FIG. 6: Fusion activity of the reconstituted viral membranes accordingto the invention. Reconstituted viral membranes containingpyrene-phospholipid were mixed with erythrocyte ghosts and fusion wasmeasured according the text.

FIG. 7: IgG titers in blood after a single intramuscular vaccination;the titers were determined 3 weeks after vaccination. Pre-immune titerswere subtracted. Vaccines used were virosomes prepared according to EP0538437, or reconstituted viral membranes, containing lipopeptides,according to the present invention. Group size was 10 mice.

FIG. 8: Equilibrium density sucrose gradient analysis of reconstitutedviral membranes from the A/Wyoming strain of virus, showing a singledenisty peak of reconstituted material; lipopeptides were recovered fromfractions 4, 5 and 6.

FIG. 9: IgG titers in blood after intranasal vaccinations on day 0 and14, in a group of 10 mice. Antigen was from the A/Panama/2007/99 strainof virus, the membranes containedN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(prolyl)₃-proline.

FIG. 10: IgA titers in nose and lung, in a group of 10 mice, after twointranasal vaccinations with reconstituted membranes of theA/Panama/2007/99 strain, containing the lipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(prolyl)₃-proline,14 days apart; the titers were determined 3 weeks after the lastvaccination. Pre-immune titers were subtracted.

EXAMPLES Example 1 Production of a Reconstituted Viral MembraneContaining the LipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,the Natural Lipids of Influenza Virus and the Influenza MembraneProteins

Influenza virus was produced by growing virus acquired from the WorldInfluenza Center or the American Type Tissue Culture Collection (ATCC),using methods known to persons skilled in the art, for instance bygrowing the virus on embryonated eggs or cultured cells. The virus wasthen purified, preferably by differential or density gradientultracentrifugation or a combination thereof, and may subsequently beinactivated by beta-propiolactone or formaldehyde according toestablished standard procedures.

The purified and concentrated influenza A/Panama/2007/9 virus (1500 nmolphospholipid) was incubated with 1 ml of the detergent octa(thyleneglycol)-n-dodecyl monoether (C12E8) (Boehringer, Mannheim, Germany) at aconcentration of 100 mM (a concentration above the detergent's criticalmicelle concentration is required), for 10 min at 4° C., in an isotonicbuffer at neutral pH: 145 mM NaCl, 2.5 mM HEPES, 1 mm EDTA, pH 7.4(Buffer A). The viral nucleocapsid and matrix proteins were then removedby centrifugation at 100,000×g for 30 min at 4° C. The pellet wasdiscarded, and the supernatant mixed with the dry lipopeptide at a ratioof 0.5 mg lipopeptide per 750 nmol of viral lipid, and mixed until thelipopeptide was dissolved. 128 mg of BioBeads SM-2 (Bio-Rad) were thenadded to each 350 microliters of the mixture, and the detergent wasremoved by shaking the mixture and the beads vigorously for one hour.The fluid was then transferred to another 64 mg of these beads andshaking was continued for 10 minutes. The resulting turbid supernatantcontains the reconstituted viral membranes, and can be used forvaccination with or without further purification.

For analysis of the physical association between the lipids,lipopeptides and viral proteins, the turbid mixture containing thereconstituted viral membranes was loaded atop a discontinuous sucrosegradient, containing a 1 mL cushion of 40% sucrose (w/v) in buffer A anda 4 mL top layer of 10% sucrose (w/v) in buffer A (as depicted in FIG.1). The gradients were centrifuged for 90 minutes at 100.000 g_(max),and samples were taken from the 40% cushion, the interface between the40% cushion and 10% top layer, and from the top. In these gradients,unincorporated viral proteins move into the cushion duringcentrifugation, lipid and lipopeptides not present in the reconstitutedmembranes move to the top of the gradient, and reconstituted viralmembranes can be found at the interface (FIG. 1). 15% of the viral lipidwas found near the top of the gradient, as was 6% of the lipopeptide.85% of the viral lipid, 94% of the lipopeptide, and 60% of the viralmembrane protein loaded on the gradient were found to be associated withthe reconstituted viral membrane band.

To analyze the lipid composition of the band, two samples ofreconstituted viral membranes, prepared according to the above protocol,or in the absence of added lipopeptide, were recovered from the 40/10%interface of sucrose gradient as described above, and extracted withCHCL3/MeoH, according to Folch et al. (1957). The extracted lipids andlipopeptides were analyzed by two-dimensional thin layer chromatography,with CHCl₃/methanol/H₂O 65/25/4 as the first eluent, followed byN-butanollacetic acid/water 2/1/1, and stained by derivatization withninhydrin stain (the plate was sprayed with 2% ninhydrin in N-butanol,and incubated at 80° C. for 10 minutes). The results were shown in FIG.2, and clearly demonstrate the physical association of the naturallipids of the virus with the lipopeptides.

Electron micrographs of the virosomes collected from the band of thegradient are shown in FIG. 3 and clearly show particles the size ofviruses, displaying the viral antigen spikes that are characteristic ofinfluenza viruses.

Example 2 Intranasal Immunization Experiments Using Reconstituted ViralMembranes Containing the LipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,the Natural Lipids of Influenza Virus and the Influenza MembraneProteins

Vaccination by intranasal application of a reconstituted viral membranecontaining the influenza virus hemagglutinin andN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,was compared to intranasal application of a standard subunit vaccine, ora virosome vaccine prepared according to EP 0538437. Balb/C mice wereimmunized by a intranasal instillation of 10 microliters of antigencontaining 5 μg of influenza proteins, on days 0, and 14. Blood sampleswere taken on day 0, 14, and 35, nasal and lung washes were collected onday 35. Several different strains of influenza virus were compared; micewere immunized with one type of strain each. Lung washes were performedby injection of 1.5 ml of PBS into the lungs via a syringe connected tothe trachea, followed by aspiration of 1 mL of fluid. Nasal washes werecollected by injecting 0.5 ml of PBS retrograde, via the trachea, intothe nasopharynx, the lavage fluid being collected at the nostrils.Debris and cellular components were immediately removed from the lavagefluids by centrifugation, and a protease inhibitor mix (chemstatin,antipain, leupeptine, pepstatin, final concentration 1 microgram/ml,from a 1000× concentrated stock solution in dry DMSO) was added, afterwhich the samples were frozen in liquid nitrogen and stored at −20 degC. until analysis. Samples were analyzed by IgA in nose and lung and IgGELISA against influenza proteins. The results are shown in FIG. 4 andFIG. 5 respectively.

Example 3 Membrane Fusion Activity of a Reconstituted Viral MembraneContaining the LipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,the Natural Lipids of Influenza Virus, Pyrene-labeledPhosphatidylcholine, and the Influenza Membrane Proteins

Purified and concentrated influenza A/Panama/2007/9 virus (1500 nmolphospholipid) was incubated with 1 ml of octa(ethylene glycol)-n-dodecylmonoether (C12E8) at a concentration of 100 mM, for 10 min at 4° C., inan isotonic buffer at neutral pH: 145 mM NaCl, 2.5 mM HEPES, 1 mm EDTA,pH 7.4 (Buffer A). The viral nucleocapsid and matrix protein were thenremoved by centrifugation at 100,000×g for 30 min at 4° C. The pelletwas discarded. The supernatant was mixed with the dry lipopeptide andpyrene-labeled phospholipid at a ratio of 0.5 mg lipopeptide and 150nmol1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphatidylcholine per750 nmol of viral lipid, and mixed until the lipopeptide andpyrene-labeled phospholipid were dissolved. 128 mg of BioBeads SM-2(Bio-Rad) were then added to each 350 microliters of the mixture, andthe detergent was removed by shaking the mixture and the beadsvigorously for one hour. The fluid was then transferred to another 64 mgof these beads and shaking was continued for 10 minutes.

For the measurement of membrane fusion, erythrocyte ghost targetmembranes were prepared from outdated red blood cell concentrates (bloodtype B, rhesus factor negative) by the method of Steck and Kant (1974)Fusion was measured at a concentration of 0.06 M of ghosts phospholipidand 1 μM of virosomal phospholipid, in a buffer containing 140 mM NaCl,15 mM sodium citrate at pH 5.1. Lipid mixing was monitored by dilutionof pyrPC. For this purpose, pyrene excimer fluorescence was measured, atexcitation and emission wavelengths of 345 nm (bandpass 2 nm) and 490 nm(bandpass 16 nm), respectively, in the presence of a 475 nm cut-offfilter in the emission beam. Background fluorescence was assessed atinfinite dilution of the probe, which was obtained by adding 35 μl of0.2 M C12E8. The changes in fluorescence were converted to extents offusion (f) by calculating f=100×(E₀−E)/(E₀−E_(y)), where E representsexcimer fluorescence at any time, and E₀ and E_(y) represent,respectively, the intensities at 490 nm at time zero and after theaddition of C12E8, both corrected for dilution effects. The results,shown in FIG. 6 clearly indicate strong fusion activity of thereconstituted membrane.

Example 4 Intramuscular Immunization Experiments Using ReconstitutedViral Membranes Containing the LipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,the Natural Lipids of Influenza Virus and the Influenza MembraneProteins, Compared to Immunization with Virosomes Prepared According toEP 0538437

25 μl of influenza antigen (5 μg of protein) was injected in the muscleof one hind leg of Balb/C mice on day 0. Blood samples were taken on day0 and 14. The A/Panama/2007/99 strain of virus was used for vaccinepreparation. Samples were analyzed by IgG ELISA against influenzahemagglutinin. The results are shown in FIG. 7.

Example 5 Physical Characterization of Functionally Reconstituted ViralMembranes Containing the A/Wyoming Membrane Proteins by EquilibriumDensity Gradient Centrifugation

Reconstituted membranes viral membranes containing the lipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysinewere prepared as described in example 1, loaded atop a 10-60% w/vsucrose gradient, en centrifuged at 50 000 rpm in a Beckman SW55 rotorfor 16 hours. In this type of gradient, lipids and lipopeptides remainat the top, while proteins migrate to the bottommost fraction. Samplesfrom the gradient were analyzed by refractometry, protein andphospholipid determination. The results, shown in FIG. 8 show thatessentially all the viral protein and most of the viral lipid co-purifyin a single peak. Also, lipopeptides were only recovered from fractions4, 5 and 6. These data indicate that the reconstituted membranes areparticles with a density of around 1.12 g/ml.

Example 6 Intranasal Immunization Experiments Using Reconstituted ViralMembranes Containing the LipopeptideN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(prolyl)₃-proline,the Natural Lipids of Influenza Virus and the Influenza MembraneProteins

Membranes were prepared from A/Panama/2007/99 as described in example 1above, and used to immunize mice as described in example 2. The ELISAIgG titers in serum, and the IgA titers in nose and lung are shown inFIGS. 9 and 10 respectively. These data indicate that the lysine andproline derivatives ofN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-result inapproximately equivalent enhancement of the immune response.

TABLE 1 Lipopeptides particularly suitable for making reconstitutedviral membranes according to the invention.N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-serineS-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-serineN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysineN-palmitoyl-S-2,3(bisoleoyloxy)-propyl-cysteinyl-seryl(lysil)₃-lysineS-2,3(bisoleoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysineN-palmitoyl-S-2,3(bismyristoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine S-2,3(bismyristoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysineN-palmitoyl-S-3(palmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysineN-palmitoyl-S-2,3 hydroxy-propyl-cysteinyl-seryl-(lysil)₃-lysineN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(prolyl)₃-proline N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(glutaminyl)₃-glutaminic acid

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1. A reconstituted viral membrane, the lipid bilayer of which comprisesa fusion protein of a virus, an amphiphilic adjuvant and, optionally, afurther antigen, whereby: (a) the lipid bilayer has a lipid compositionthat is compatible with fusion, induced by the fusion protein, of theviral membrane with the membrane of a cell that can be fused with thevirus from which the fusion protein is derived; (b) the fusion proteinand the amphiphilic adjuvant are incorporated within the hydrophobicinterior of the lipid bilayer; and, (c) the fusion protein, theamphiphilic adjuvant and the optional further antigen are not covalentlylinked; and wherein the amphiphilic adjuvant is a lipopeptide.
 2. Areconstituted viral membrane according to claim 1, wherein the lipidbilayer comprises natural lipids of a viral membrane.
 3. A reconstitutedviral membrane according to claim 1, wherein the amphiphilic adjuvant isa ligand for a mammalian Toll-like receptor.
 4. A reconstituted viralmembrane according to claim 1, wherein the lipopeptide is selected fromthe group consisting of:N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-serine,S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-serine,N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,N-palmitoyl-S-2,3(bisoleoyloxy)-propyl-cysteinyl-seryl-(lysil)3-lysine,S-2,3(bisoleoyloxy)-propyl-cysteinyl-seryl-serine-(lysil)₃ -lysine,N-palmitoyl-S-2,3(bismyristoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,S-2,3(bismyristoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,N-palmitoyl-S-3 (palmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysineand N-palmitoyl-S-2,3hydroxyl-propyl-cysteinyl-seryl-(lysil)₃lysine,N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(prolyl)₃-proline,N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(glutaminyl)₃-glutaminicacid.
 5. A reconstituted viral membrane according to claim 1, wherein atleast one of a fusion protein of a virus or a further antigen is anintegral membrane protein.
 6. A reconstituted viral membrane accordingto claim 1, wherein at least one of a fusion protein of a virus or afurther antigen is a viral antigen.
 7. A reconstituted viral membraneaccording to claim 6, wherein at least one of a fusion protein of avirus or a further antigen is from an influenza virus.
 8. Areconstituted viral membrane according to claim 7, wherein at least oneof a fusion protein of a virus or a further antigen is a hemagglutinin(HA), a neuraminidase (NA) or an M2 protein.
 9. A reconstituted viralmembrane according to claim 6, wherein at least one of a fusion proteinof a virus or a further antigen is derived from a virus selected fromthe group consisting of Retroviridae, rubellavirus, Paramyxoviridae,Flaviviridae, Herpesviridae, Bunyaviridae, Arenaviridae, Hantaviridae,Coronaviridae, Papovaviridae, Rhabdoviridae, Coronaviridae,Alphaviridae, Arteriviridae, Filoviridae, Arenaviridae, poxviridae, andAfrican swine fever virus.
 10. A reconstituted viral membrane accordingto claim 1, wherein the further antigen is derived from a parasite, abacterium, a fungus, a yeast, or wherein the further antigen is atumor-specific antigen.
 11. A pharmaceutical composition comprising areconstituted viral membrane as defined in claim 1 and apharmaceutically acceptable carrier.
 12. A pharmaceutical compositionaccording to claim 11, whereby the composition is suitable forintranasal, oral or parental administration.
 13. A reconstituted viralmembrane according to claim 2, wherein the amphiphilic adjuvant is aligand for a mammalian Toll-like receptor.
 14. A reconstituted viralmembrane according to claim 2, wherein at least one of a fusion proteinof a virus or a further antigen is an integral membrane protein.
 15. Areconstituted viral membrane according to claim 3, wherein at least oneof a fusion protein of a virus or a further antigen is an integralmembrane protein.
 16. A reconstituted viral membrane according to claim4, wherein at least one of a fusion protein of a virus or a furtherantigen is a hemagglutinin (HA), a neuraminidase (NA) or an M2 protein.17. A reconstituted viral membrane according to claim 16, wherein thefusion protein of a virus is a hemagglutinin (HA) and the furtherantigen is a neuraminidase (NA).